New software speeds targeted TB gene sequencing

Drug-resistant tuberculosis poses a major challenge because clinicians need fast, accurate information about which mutations are present in an infection to choose the right treatment. Amplicon sequencing (Amp-Seq) — a targeted way to read specific Mycobacterium tuberculosis genes — is a cost-effective option for profiling infections and guiding care, but these tests must be updated continuously as new resistance mutations and loci are discovered. To tackle that need, Jody Phelan and colleagues developed TOAST (Tuberculosis Optimised Amplicon Sequencing Tool), a customisable software tool that helps researchers and clinicians design targeted amplicons. TOAST is informed by an integrated and growing database of mutations drawn from more than 50K M. tuberculosis isolates. The software lets users set practical lab parameters such as melting temperature, amplicon length and GC content, while also checking for problematic primer behaviors. By automating much of the design work and tying it to a large mutation database, TOAST aims to make it simpler and faster to update Amp-Seq assays so those tests remain relevant as the genetics of resistance change.

TOAST is built to optimise amplicon design across any loci and for multiple sequencing platforms, including Illumina and Oxford Nanopore Technology (ONT). The tool checks for primer interactions such as homodimer formation and non-specific binding so that designed primers work reliably in the lab. To demonstrate how it performs, the team used TOAST to design a set of 33 amplicons in a single multiplex group, with priority given to covering resistance-associated mutations — including insertions, deletions and single nucleotide polymorphisms (SNPs) — across 13 different drugs. The researchers also established an experimental protocol compatible with ONT sequencing and validated it on two clinical samples known to have multi-drug resistance. Sequencing these samples produced a minimum depth coverage exceeding 50-fold for each amplicon region, showing that the design and lab workflow produced robust, high-coverage data for targeted regions.

The combination of a purpose-built design tool and a validated lab protocol means TOAST could speed the development of new Amp-Seq assays for detecting drug-resistant TB. Because the software is customisable and linked to a large and expanding mutation database, labs can update assays to include newly discovered loci or mutations without starting from scratch. Compatibility with both Illumina and Oxford Nanopore Technology (ONT) platforms gives flexibility for different budgets and settings, and the checks for primer quality reduce the chance of wasted lab work. The authors argue that these features — plus the demonstrated ability to get deep coverage from a single multiplexed panel — make TOAST a practical option for developing Amp-Seq tests that inform treatment decisions, improve patient outcomes, and help curb the spread of resistant infections, especially where cost and adaptability matter most.